In the mitochondria of Trypanosoma brucei, RNAs are synthesized polycistronically. Nevertheless, levels of mature monocistronic RNAs vary dramatically between life cycle stages. This indicates that steady-state RNA abundance, and thus gene expression, is controlled by posttranscriptional processes and these processes are developmentally regulated. Our hypothesis is that RNA stability plays a critical role in the regulation of gene expression in this system. The goal of this proposal is to elucidate the mechanism by which mitochondrial RNA stability is regulated, and to understand the role played by this process in gene regulation. Using an in organello system developed in our laboratory, we have identified a novel UTP-stimulated rapid pathway for degradation of polyadenylated RNA. In Aim 1, we will further exploit this in organello system to determine the mechanism by which UTP stimulates poly(A)+ RNA degradation. The in organello system will also be used to define the direction of RNA degradation, determine whether degradation is exclusively exonucleolytic, and elucidate the role of mRNA poly(A) tails in determining degradation rate or pathway. Degradation rates of specific RNAs will be compared in procyclic and bloodstream form mitochondria to directly address the biological significance of regulated mRNA stability in this system. In Aim 2, we will develop an in vitro RNA degradation system. This system will be used to identify degradation intermediates and dissect the degradation pathway. The roles of cis-acting RNA sequences, 5' and 3' end moieties, and 3' tail length and composition will be determined. In Aim 3, we will use a three-pronged approach to purify protein factors involved in the RNA degradation process. The in vitro assay will be utilized as a starting point for purification of multicomponent degradation complexes. We will also focus on identification and isolation of exoribonucleases. Finally, we will use genetic and biochemical means to isolate T. brucei homologs of two proteins that function in RNA degradation in other systems, polynucleotide phosphorylase and RNase E. These experiments will elucidate the biochemical features of mitochondrial mRNA degradation in T. brucei, and provide information on the role of this process in gene expression. Moreover, since little is known about RNA degradation in the mitochondria of any system, these studies will provide insights into the process of regulated mitochondrial RNA stability in higher organisms.